Monoclonal antibodies that suppress B cell growth and/or differentiation

ABSTRACT

The present invention provides monoclonal antibodies which interfere with the interactions between FDCs and B cells, thereby suppressing the proliferation and/or differentiation of B cells in lymphoid follicles. The monoclonal antibodies of the present invention are useful for treating follicular lymphomas, multiple myeloma as well as autoimmune diseases.

FIELD OF THE INVENTION

[0001] This invention relates to monoclonal antibodies which affectgrowth and/or differentiation of B cells in the germinal center. Themonoclonal antibodies of the present invention are useful for treatingdisorders characterized by an abnormal growth and/or differentiation ofB cells, including lymphoma, multiple myeloma and autoimmune diseases.

DESCRIPTION OF RELATED ART

[0002] As B cells mature, B cells leave the bone marrow and migrate tothe lymphoid follicles of lymph nodes and spleen, and other peripherallymphoid tissues. In the absence of antigen, mature B cells pass fromblood into primary lymphoid follicles and then back into peripheralblood. If B cells encounter antigens and appropriate helper T-cells onentering the lymphoid tissue and become activated, B cells proliferatefirst in the T-cell areas, forming primary foci from which proliferatingB cells migrate to the primary follicle forming a secondary folliclewith a germinal center (or “GC”).

[0003] The GC of the secondary lymphoid follicles is a unique automicalsite where antigen-activated B cells undergo clonal expansion andselection to differentiate into memory B cells or intoantibody-secreting plasma cells. Follicular dendritic cell “FDC” is astromal cell located in the GC which is essential for B cell growth andlymphomagenesis. The GC reaction is initiated by rapid proliferation ofAg-stimulated B cells in association with follicular dendritic cells or“FDCs” (MacLennan, I. C., Annu. Rev. Immunol. 12:117-139, 1994). TheGC-B cells exhibit features distinct from naive or memory B cells inthat GC-B cells display a unique pattern of Ag expression on the cellsurface (Liu, Y. J., et al., Immunity 2:239-248, 1995), undergo Agreceptor-mediated apoptosis (Billian, G., P., et al., Eur. J. Immunol.27:405-414, 1997), and require essential survival signals from FDCs, asdisruption of FDC-B cell clusters results in apoptosis of B cells(Kosco, M. H., et al., J. Immunol. 148:2331-2339, 1992; Koopman, G., etal., J. Immunol. 152:3760-3767, 1994). Observations made usinglymphotoxin-α knockout mice further confirmed that the initialinteraction between FDCs and B cells is essential for GC formation(Gonzalez, M., F., et al., J. Exp. Med. 187:997-1007, 1998; Fu, Y. -X.,et al., J. Exp. Med. 187:1009-1018, 1998). T cells expressing CD40ligand (CD40L) also play a pivotal role in the GC reaction, as evidencedin hyper-IgM patients and in mouse models that have null mutations inthe CD40 (Kawabe, T. et al., Immunity 1:167-178, 1994) or CD40L genes(Renshaw, B. R., et al., J. Exp. Med. 180:1889-1900, 1994).

[0004] The genetic events that occur during the clonal expansion andselection of B cells at the GC include somatic mutation and isotypeswitching, which leads to the production of more efficient antibodieswith high affinity to the invading microorganisms. At the same time, Bcell lymphoma may arise in the GC of the secondary lymphoid follicles asa consequence of genetic instability and mobility during this cellularand molecular process.

[0005] Follicular lymphoma is the most common type of non-Hodgkin'slymphoma in the west. In the early stage, follicular lymphoma is usuallyindolent, regressing spontaneously and showing susceptibility tochemotherapy (Horning, S. J., et al., N. Engl. J. Med. 311:1471-1475,1984). However, this tumor usually recurs and can undergo blasttransformation to an aggressive form, ultimately becoming a fataldisease. The generation and blast transformation of this tumor isclosely associated with FDCs in the GC (Petrasch, S., et al., Br. J.Haematol. 80:21-26, 1992).

[0006] Multiple myeloma is a tumor usually found in bone marrow. Myelomacells grow aggressively and, like plasma cells (PCs) from which myelomacells are believed to originate, secrete immunoglobulins. PCs aregenerated in the GC as a result of clonal expansion and selection,during which process somatic mutation and isotype switching occur. PCsthen migrate to bone marrow to expand in the presence of stroma cells.As a consequence of the genetic mobility and mutability involved in thegeneration and translocation of PCs, malignant transformation may occurwhich leads to the development of multiple myeloma.

[0007] The present invention provides monoclonal antibodies whichinterfere with the interactions between FDCs and B cells, therebysuppressing the growth and/or differentiation of B cells, as well astumorigenesis of B cells in vivo. The monoclonal antibodies of thepresent invention are useful for treating disorders characterized by anabnormal growth and/or differentiation of B cells, including lymphoma,multiple myeloma and autoimmune diseases.

SUMMARY OF THE INVENTION

[0008] One embodiment of the present invention provides monoclonalantibodies which suppress the growth and/or differentiation of B cellsin the lymphoid follicles.

[0009] A preferred monoclonal antibody of the present invention is mAb8D6. The hybridoma cell line which produces mAb 8D6 was deposited onMar. 13, 2001 with the American Type Culture Collection, 10801University Boulevard, Manassas, Va. 20110-2209 (ATCC#).

[0010] Another preferred monoclonal antibody of the present invention ismAb 4G10. The hybridoma cell line which produces mAb 4G10 was depositedon Mar. 13, 2001 with the American Type Culture Collection, 10801University Boulevard, Manassas, Va. 20110-2209 (ATCC#).

[0011] Hybridoma cell lines which produce mAb 8D6 and mAb 4G10,respectively, are provided in another embodiment of the presentinvention.

[0012] Functional derivatives of the monoclonal antibodies of thepresent invention are also provided, including, but not limited to, Fab,Fab′, F(ab′)₂, single chain antibodies, chimeric antibodies and thelike.

[0013] Another embodiment of the present invention is directed topharmaceutical compositions. The pharmaceutical compositions include amonoclonal antibody of the present invention or a functional derivativethereof, and a pharmaceutically acceptable carrier.

[0014] A further aspect of the invention provides methods of treating asubject suffering a pathological condition characterized by abnormal Bcell growth or differentiation by administering to the subject atherapeutically effective amount of a monoclonal antibody of the presentinvention or a functional derivative thereof. Pathological conditionswhich can be treated by practicing the present methods include lymphoma,multiple myeloma and autoimmune diseases.

BRIEF DESCRIPTION OF DRAWINGS

[0015] FIGS. 1A-1F depict immunohistological localization of 8D6 Agexpression in human FDCs. The serial cryostat sections from a normalhuman tonsil (C and D) or cytospin preparations of human tonsil FDCclusters (E and F) were stained with mAbs 8D6 (A, C, and E), DRC-1 (Band D), and 7D6 (F). A and B are higher power views of boxed areas in Cand D, respectively, showing the network staining pattern. Originalmagnifications: (C and D)×100; (E and F)×1,000.

[0016] FIGS. 1G-1H depict the expression of 8D6 Ag on HK cells. HK cellswere stained with mAbs 8D6 (G, filled histogram), 3C8 (H, filledhistogram), or isotype control mouse IgG1 (G and H, open histograms),followed by FITC-conjugated goat anti-mouse IgG. FITC intensity wasanalyzed by FACScan™.

[0017] FIGS. 2A-2C depict inhibition of FDC-B cell interaction by mAb8D6. (2A) Tonsillar B cells were cocultured with irradiated FDC clusters(2,000 clusters per well; 5,000 rads) for 10 d in the presence of mAb8D6 or 3C8, anti-CD40 (100 ng/ml), rhIL-2 (10 U/ml), and rhIL-10 (30ng/ml). IgG levels in conditioned media were measured by humanIgG-specific ELISA. IgG concentrations in the control cultures with Bcells and B cells plus FDCs were 0.26 and 1.8 mg/ml, respectively. (2Band 2C) GC-B cells were cocultured with or without mAb 8D6 in thepresence of irradiated HK cells, CD40L, rhIL-2, and rhIL-10. Results ofviable cell recovery (2B, left), IgG secretion (2B, right), and cellcycle progression (2C) are shown.

[0018] FIGS. 3A-3C depict the inhibition by mAb 8D6 of HK-dependentL3055 cell proliferation. HK-dependent L3055 cells (2×10⁴ cells/ml) werecultured in the presence of irradiated HK or CD32-transfected L cells(2×10⁴ cells per well; 5,000 rads) for the indicated time periods in24-well plates (3A). In a separate experiment (3B), L3055 cells (2×10⁴cells/ml) were cultured for 5 days with irradiated HK cells (2×10⁴ cellsper well; 5,000 rads) pretreated with culture medium alone (control),mAb 8D6, mAb 4G10, or a combination of mAb 8D6 and mAb 4G10 (20 μg/mleach). Viable cells were counted by trypan blue exclusion assay, andviable cell recovery percentages are shown. (3C) L3055 cells (10⁴cells/ml) were cultured in the presence or absence of irradiated HKcells for 24 h. HK cells were pretreated with mAbs 8D6 or 3C8 forblocking experiments. Cultured cells were stained with FITC-labeledannexin V and propidium iodide.

[0019] FIGS. 4A-4C depict stimulation of B cell proliferation anddifferentiation by 8D6 Ag cDNA-transfected COS cells. Mock (COS_(Mock))or 8D6 Ag cDNA (COS_(8D6))-transfected COS cell-B cell cocultures wereperformed. The viable cell number of B cells (4A) and IgG secretion data(4B) was shown. In blocking experiments, transfected COS cells weretreated with mAb 8D6 for 30 min at 37° C. before coculture with B cells(4C).

[0020] FIGS. 5A-5B depict lymphoma formation in the presence of HKcells. Nude mice were subcutaneously (s.c.) injected using L3055 cellswithout (5A) or with (5B) HK cells. Images were taken 60 days afterinoculation.

[0021] FIGS. 6A-6C depict the inhibition of lymphoma formation in nudemice by FDC-binding mAbs. 6A. L3055 cells and FDC-binding mAbspretreated HK cells were inoculated into nude mice. The size of solidtumor was measured twice a week with a caliber and expressed in volumn(volumn=length×width×height, in mm³). A tumor growth curve of a typicalexperiment was shown. 6B. A summary of in vivo xenoraft experiments. Thenumber of experiment and the size of tumor formed on day 60 were shown.6C. A photo of nude mice inoculated as described in 6A at day 38. Cellsand mAbs injected were as follows: 1, HK+L3055; 2, HK+L3055+8D6; 3,HK+L3055+4G10; and 4, HK+L3055+8D6+4G10.

[0022] FIGS. 7A-7D depict the stimulation of plasma cell (PC) generationby FDC-signaling molecule-8D6 (FDC-SM-8D6).

[0023]FIG. 8 depicts the differentiation of centrocytes into plasmacells (PC) in the presence of IL-10. Centrocytes were generated byculturing GC-B cells for 5 days in the presence of HK cells, CD40L, IL-2and IL-4. The cells were washed and re-cultured with IL-4 or IL-10 for 7additional days in the presence of HK cells, CD40L, and IL-2. At the endof culture, the harvested cells were stained with anti-CD20 andanti-CD38 mAbs for FACS analysis. Percentages of PCs ( CD20⁻CD38^(hi))or memory B cells (CD20⁺CD38^(lo)) are indicated by the numbers adjacentto their gates.

[0024]FIG. 9 depicts the inhibition by mAb 8D6 of PC generation fromcentrocytes in culture with IL-10. GC-B cells were cultured in two stepsas described in Example 7. Viable cell number and IgG secretion assessedat the end of both culture steps without mAb (open), with mAb 8D6(black) or control mAb 3C8 (hatched) are shown.

DETAILED DESCRIPTION OF THE INVENTION

[0025] One embodiment of the present invention is directed to monoclonalantibodies which suppress the growth and/or differentiation of B cellsin the lymphoid follicles.

[0026] Without intending to be limited to any particular theory, theinhibitory effects of the instant monoclonal antibodies are believed tobe achieved by interfering with the interactions between folliculardendritic cells (FDCs) and B cells, as the proliferation anddifferentiation of B cells in the lymphoid follicles depend oninteractions of B cells with FDCs. The term “interaction” encompassesinteractions mediated by direct cell-cell contact, as well asinteractions mediated by molecules secreted by FDCs that directly orindirectly affect the proliferation and differentiation of B cells inthe lymphoid follicles.

[0027] The monoclonal antibodies of the present invention can begenerated essentially as follows. An animal (e.g., mouse, sheep orrabbit) is immunized with isolated human FDCs. The animal is preferablyneonatally tolerized with human tonsillar mononuclear cells (MNCs) priorto the immunization with human FDCs. Spleen cells are isolated from theimmunized animal and used in fusion with myeloma cells for makinghybridomas. Hybridomas that make monoclonal antibodies capable ofinhibiting FDC-dependent B cell growth or differentiation are thenidentified and cloned.

[0028] Preferred monoclonal antibodies of the present invention are mAb8D6 and mAb 4G10. The hybridoma cell lines that produce mAb 8D6 (ATCC #)and mAb 4G10 (ATCC#), respectively, were deposited on Mar. 13, 2001 withAmerican Type Culture Collection (ATCC#).

[0029] Hybridoma cell lines which produce mAb 8D6 and mAb 4G10 formanother embodiment of the present invention.

[0030] These hybridomas represent preferred sources for preparing mAb.8D6 and mAb 4G10. Monoclonal antibodies can be purified from culturesupernatant of the hybridomas grown in tissue culture flasks.Alternatively, hybridoma cells can be injected into animals to produceinflammatory ascites. Antibody-containing ascites can be harvested 8-12days after intraperitoneal injection. The ascites contain a higherconcentration of antibodies, but include both monoclonals andimmunoglobulins from the animal.

[0031] Monoclonal antibodies in the hybridoma supernatant or in theascites can be purified by a variety of conventional proteinpurification methods, for example, affinity chromatography.Alternatively, hybridoma supernatant or the ascites can be used directlyin place of the monoclonal antibodies under certain circumstances. Thoseskilled in the art can determine whether purification of the monoclonalantibodies is required for a particular use.

[0032] Another embodiment of the present invention is directed tofunctional derivatives of the monoclonal antibodies of the presentinvention. “Functional derivatives” refer to antibody molecules orfragments thereof which are derived from a monoclonal antibody of thepresent invention and which have retained the antigenic specificity andthe functional activity (i.e., inhibiting FDC-dependent proliferationand/or differentiation of B cells) of the original monoclonal antibody.Examples of functional derivatives include Fab, Fab′, F(ab′)₂ of thepresent mAbs, single chain antibodies, chimeric antibodies and the like.

[0033] Techniques which can be employed for making Fab, Fab′, andF(ab′)₂ fragment of an antibody are well known in the art. See, e.g.,Coligan et al. Current Protocols in Immunology, John Wiley & Sons Inc.,New York, N.Y. (1994); Harlow, E. and Lane, D., Antibodies: A LaboratoryManual, Cold Spring Harbor Press, 1988.

[0034] A single-chain antibody (sAb) is created by fusing together thevariable domains of the heavy and light chains using a short peptidelinker, thereby reconstituting an antigen-binding site on a singlemolecule. Such single-chain antibody variable fragments (Fvs) can befused to all or a portion of the constant domains of the heavy chain ofan immunoglobulin molecule, if necessary. The use of sAb avoids thetechnical difficulties in the introduction of more than one geneconstruct into host cells. Single chain antibodies and methods for theirproduction are known in the art. See, e.g., Bedzyk et al. J. Biol.Chem., 265:18615 (1990); Chaudhary et al. Proc. Natl. Acad. Sci.,87:9491 (1990); U.S. Pat. No. 4,946,778 to Ladner et al.; and U.S. Pat.No. 5,359,046 to Capon et al.

[0035] Chimeric antibodies as used herein refer to antibodies whichcontain the antigen-binding portion of the murine monoclonal antibodiesof the present invention and a portion of an immunoglobulin from anotherspecies. For example, a chimeric antibody of the present invention cancontain the variable region of mAb 8D6, joined to the constant region ofa human immunoglobulin. Chimeric antibodies and methods for theirproduction are known in the art. See, e.g., Cabilly et al., EuropeanPatent Application 125023 (published Nov. 14, 1984); Taniguchi et al.,European patent Application 171496 (published Feb. 19, 1985); Morrisonet al., European Patent Application 173494 (published Mar. 5, 1986);Neuberger et al., PCT Application WO 86/01533, (published Mar. 13,1986); Kudo et al., European Patent Application 184187 (published Jun.11, 1986); Robinson et al., International Patent Publication#PCT/US86/02269 (published May 7, 1987); Liu et al., Proc. Natl. AcadSci. USA 84:3439-3443 (1987); Sun et al., Proc. Natl. Acad. Sci. USA84:214-218 (1987); Better et al., Science 240:1041-1043 (1988). Thesereferences are incorporated herein by reference. Generally, DNA segmentsencoding the H and L chain antigen-binding regions of the murine mAb canbe cloned from the mAb-producing hybridoma cells, which can then bejoined to DNA segments encoding C_(H) and C_(L) regions of a humanimmunoglobulin, respectively, to produce murine-human chimericimmunoglobulin-encoding genes. Humanized chimeric antibodies can also bemade by constructing a reshaped human antibody, which has been describedin, e.g., Maeda et al., Hum. Antibod. Hybridomas 2: 124-134 (1991), andPadlan, Mol. Immunol. 28: 489-498 (1991).

[0036] A derivative of the monoclonal antibodies of the presentinvention can be tested to determine whether such derivative possessesthe antigenic specificity and the functional activity of the originalmonoclonal antibody. Antigenic specificity can be assessed in assays,e.g., staining of FDCs or HK cells, or Western Blot analysis usingFDC-SM-8D6. The functional activity of a derivative can be assessed inassays, e.g., coculturing GC-B cells with FDCs or HK cells with orwithout the derivative in the presence of CD40L, rIL2, rIL10 andassaying for B cell growth and/or differentiation.

[0037] A further aspect of the invention is directed to pharmaceuticalcompositions which include a monoclonal antibody of the presentinvention, or a functional derivative thereof, or combinations thereof.Preferred monoclonal antibodies for use in the pharmaceuticalcompositions of the present invention are mAb 8D6 and mAb 4G10.

[0038] The pharmaceutical compositions of the present invention caninclude other substances such as cytokines, adjuvants andpharmaceutically acceptable carriers. As used herein, a pharmaceuticallyacceptable carrier includes any and all solvents, including water,dispersion media, culture from cell media, isotonic agents and the likethat are non-toxic to the host. Preferably, it is an aqueous isotonicbuffered solution with a pH of around 7.0. The use of such media andagents in therapeutic compositions is well known in the art.

[0039] A further aspect of the invention is directed to methods oftreating subject suffering a pathological condition characterized byabnormal growth or differentiation of B cells. In accordance with thepresent invention, the subject is treated by administration of atherapeutically effective amount of an antibody of the presentinvention.

[0040] By “treating” is meant that the abnormal growth ordifferentiation of B cells is inhibited, reduced, or eliminated, or theoccurrence of the abnormality of B cell growth or differentiation isprevented or delayed.

[0041] A “subject” which can be treated in accordance with the presentmethods can be any mammalian subject, including humans, dogs, monkeys,cows and the like. A preferred subject to be treated is a humanindividual.

[0042] Pathological conditions which can be treated in accordance withthe present invention include lymphoma, multiple myeloma and autoimmunediseases such as rheumatoid arthritis, systemic lupus erythematous andmultiple sclerosis.

[0043] Preferably, the present method is employed to treat patientssuffering lymphoma, including both Hodgekin's and non-Hodgekin'slymphoma that are follicular lymphoma or diffuse large B cell lymphoma.These lymphomas are most common type of lymphomas in adult in the West.

[0044] Preferred antibodies for use in the administration include mAb8D6, mAb 4G10, a functional derivative of such monoclonal antibodies, ora combination thereof. More preferably, a combination of mAb8D6 and mAb4G10, or a combination of functional derivatives (e.g., humanizedantibodies) of mAb8D6 and mAb 4G10, is administered to the subject.

[0045] An antibody can be administered alone or together with apharmaceutically acceptable carrier. A pharmaceutically acceptablecarrier includes all solvents, such as fats, oils, water, salinesolutions, lipids, liposomes, resins, binders, fillers, dispersionmedia, cell culture media, and the like, or combinations thereof, thatare non-toxic to the recipient subject.

[0046] In accordance with the present invention, an antibody or anantibody derivative can be combined with the carrier in any convenientand practical manner, e.g., by solution, suspension, emulsification,admixture, encapsulation, absorption and the like, and if necessary, byshaping the combined compositions into pellets or tablets. Suchprocedures are routine for those skilled in the art.

[0047] Dosages of an antibody or an antibody derivative to betherapeutically effective depend on the disease state and other clinicalfactors, such as weight and condition of the subject, the subject'sresponse to the therapy, the type of formulations and the route ofadministration. The precise dosage of an antibody to be therapeuticallyeffective can be determined by those skilled in the art. As a generalrule, the therapeutically effective dosage of an antibody can be in therange of about 0.5 μg to about 2 grams per unit dosage form, orpreferably, about 0.5 μg to about 1 mg per unit dosage form. A unitdosage form refers to physically discrete units suited as unitarydosages for mammalian treatment: each unit containing a pre-determinedquantity of the active material calculated to produce the desiredtherapeutic effect in association with any required pharmaceuticalcarrier. The methods of the present invention contemplate single as wellas multiple administrations, given either simultaneously or over anextended period of time.

[0048] The administration of an antibody may be carried out in anyconvenient manner, including by aerosol inhalation, injection,ingestion, transfusion, implantation or transplantation. Preferably, theantibodies of the present invention are administered to a patient byinjection, and more perferably subcutaneous (s.c.), intraperitoneal(i.p.), intra-arterial (i.a.), or intravenous (i.v.) injection.Preferably, the injection is near the tumor site if possible.

[0049] All the publications mentioned in the present disclosure areincorporated herein by reference. The terms and expressions which havebeen employed in the present disclosure are used as terms of descriptionand not of limitation, and there is no intention in the use of suchterms and expressions of excluding any equivalents of the features shownand described or portions thereof, it being recognized that variousmodifications are possible within the scope of the invention.

[0050] The present invention is further illustrated by the followingexamples.

EXAMPLE 1

[0051] Antibodies

[0052] Abs used in the following examples were anti-CD40 (G28-5;American Type Culture Collection); DRC-1 (clone R4/23; DAKO,Carpinteria, Calif.); 7D6 (from. Dr. Yong-Jun Liu, Laboratory forImmunological Research, Schering-Plough Corp., Dardilly, France);isotype matched control mAb 3C8 (IgG₁) (Choe et al., J. Immunol.157:1006, 1996); biotin-conjugated goat anti-mouse Ig or horseradishperoxidase-conjugated goat anti-human IgG (BioSource International,Camarillo, Calif.); streptavidin-biotinylated horseradish peroxidasecomplex (Amersham Life Science, Piscataway, N.J.); FITC-conjugated goatanti-mouse IgG (PharMingen); rabbit anti-human IgG or alkalinephosphatase-conjugated rabbit anti-mouse IgG (ICN Biomedi-cals);horseradish peroxidase conjugated goat anti-human IgG (Cappelpharmaceutical, Inc. Malvern, Pa.); anti-CD44 (NKI-P1, IgG₁, Dr. C. G.Figdor, University Hospital Nijmegen, Nijmegan, The Netherlands); ratanti-mouse IgG₁, microbeads (Miltenyi Biotech Inc., Sunnyvale, Calif.);and FITC-conjugated anti-CD20, PE-conjugated anti-CD38, andFITC-conjugated goat anti-mouse Ig (Pharmingen, San Diego, Calif.).

[0053] Cell Lines, Cytokines and Reagents

[0054] Tonsillar B cells were prepared and GC-B cells were isolated bymagnetic cell separation (MACS; Miltenyi Biotec) as described by Choe etal. (J. Immunol. 157:1006-1016, 1996). Fresh FDC clusters were isolatedfrom tonsils of 3-10-yr-old children as described by Choe et al. (J.Immunol 164:56-63, 2000). The FDC line, HK, was established andmaintained as described by Kim et al. (J. Immunol. 155:1101-1109, 1995).The L3055 cell line was cocultured with HK cells in IMDM (IrvineScientific) supplemented with 10% FCS (Life Technologies), 2 mMglutamine, 100 U/ml penicillin G, and 100 mg/ml streptomycin (IrvineScientific). GC-B cells were culture in Iscove's modified Dulbecco'smedium (Irvine Scientific, Santa Ana, Calif.) supplemented with 10%fetal calf serum (Life Technologies, Inc., Grand Island, N.Y.), 2 mMglutamine, 100 U/ml penicillin G, and 100 μg/ml streptomycin (IrvineScientific).

[0055] Annexin V-FITC apoptosis detection kit was purchased fromTrevigen. Cytokines used in the following examples were CD40L (ImmunexCorp.), recombinant human (rh)IL-2 (Hoffman-La Roche), rhIL-10 (R&DSystems), rhIL-4 (Schering-Plough Corp.), and rhIL-6 (Sandoz ResearchInstitute).

EXAMPLE 2 Generation of mAbs Staining FDCs

[0056] Murine mAbs that react to human tonsillar FDCs were generatedusing a procedure that involved tolerization before immunization(Golumbeski et al., Anal. Biochem. 154:373-381, 1986). Tolerization wasachieved by injection of newborn (within 40 h after birth) BALB/c micewith tonsillar mononuclear cells (MNCs) containing human T and B cells.At 2 month of age, this animal was injected intraperitoneally withfreshly isolated FDC clusters (2×10⁴) three times in a 2-week interval.After the third immunization, the serum from the immunized mouse showedstrong reactivity to tonsillar FDCs in frozen tissue sections, but noreactivity to MNCs as assayed by the cell-based ELISA. Spleen cells fromthis mouse were used as fusion partners with a mouse myeloma cell line,SP 2/0, to generate mAbs. After the cell fusion, more than 600hybridomas were grown, and their supernatants were screened for FDCstaining as follows. The adjacent cryostat sections of a human tonsilwere fixed in cold acetone. Slides were blocked with 1% (wt/vol)BSA-PBS, then incubated with supernatants from hybridomas, followed byFITC-conjugated goat anti-mouse Ig, then observed under the fluorescencemicroscope. The hybridoma supernatants containing more than 10 mg/ml ofmouse Ig were subjected to ELISA which tested for reactivities to MNCs.The hybridomas reacting to MNCs were discarded. The hybridomas that werenegative in MNC-based ELISA were subjected to the next screening step:HK binding by ELISA and immunohistochemical staining of the frozensections of the tonsillar tissues. These screening steps were repeatedduring a limiting dilution procedure to select a single mAb-producingclone.

[0057] Twenty-eight (28) hybridomas were identified that specificallystained the tonsillar GC. Of 28 hybridomas, 17 mAb clones were obtainedby a limiting dilution method. 16 IgG mAbs were purified from ascites byaffinity chromatography with protein A/G. One of these mAbs, an IgG1called 8D6, recognized a molecule expressed in human tonsillar GCs (FIG.1, A and C) with an immunohistochemical staining pattern similar to thatof the known FDC-specific mAb, DRC-1 (FIGS. 1, B and D). Monoclonal AbDRC-1 has been described by Naiem, M., et al. (J. Clin, Pathol.36:167-175, 1983). The mAb 8D6 staining was restricted to the follicles(FIG. 1C). There is a clear demarcation between the GC and the Tcell-rich area outside of the GC, which is negative for all three mAbs(e.g., DRC-1, 7D6, and 8D6). The 8D6 Ag is abundantly expressed in theGC, staining the reticular network in the higher magnification (FIG.1A). The diffuse staining pattern of mAb 8D6 in the GC is characteristicof FDCs surrounding MNCs in the follicles. Such a staining pattern wasconfirmed by the simultaneous staining of the adjacent tissue sectionswith known FDC-specific mAbs such as DRC-1 (FIG. 1B) and 7D6. mAb 7D6has been described by Liu et al. (J. Exp. Med. 185:165-170, 1997). Asingle cell suspension was prepared as a cytospin. The cytospinpreparations were stained with FDC-staining mAbs and examined under themicroscope. At the single cell level, mAbs 8D6 (FIG. 1E) and 7D6 (FIG.1F), both stained large, cytoplasm-rich, sometimes binucleated FDCs. Theisotype-matched control Abs did not stain FDCs, excluding thepossibility of autofluorescence.

[0058] Another mAb of the 16 mAbs, 4G10, also specifically stained FDCsin the GC. Both mAb 8D6 and mAb 4G10 showed positive binding to HK cellswhen analyzed by FACS.

EXAMPLE 3 Functional Blocking Activity of FDC-Specific mAb 8D6 in FDC-BCell Interaction

[0059] To measure its ability to block FDC-B cell interaction, mAb 8D6was used in the coculture of B cells and FDC clusters. BecauseAg-activated T cells participate in GC reactions by direct cell-to-cellcontact and by secreting cytokines (Han, S., et al., J. Immunol.155:556-567, 1995), the defined signals of activated T cells, such asanti-CD40, IL-2 and IL-10, were used to characterize mAb 8D6 functions.

[0060] Tonsillar B cells were cocultured with irradiated FDC clusters(2,000 clusters per well; 5,000 rads) for 10 days in the presence of mAb8D6 or 3C8, anti-CD40 (100 ng/ml), rhIL-2 (10 U/ml), and rhIL-10 (30ng/ml). IgG levels in conditioned media were measured by humanIgG-specific ELISA. IgG concentrations in the control cultures with Bcells and B cells plus FDCs were 0.26 and 1.8 mg/ml, respectively. Ascan be seen from FIG. 2A; mAb 8D6 consistently inhibited theFDC-mediated B cell IgG secretion. The blocking activity of mAb 8D6 isspecific because another FDC-specific murine IgG1, mAb 3C8, did notinhibit the FDC costimulatory activity (FIG. 2A). Monoclonal Ab 3C8, asdescribed by Choe et al. (J. Immunol. 157:1006-1016, 1996), was preparedby immunizing mice with HK cells, and its Ag was abundantly expressed inFDC and HK cells (FIG. 1H). In addition to mAb 3C8, the other 13 IgG1,FDC-specific mAbs prepared in parallel with mAb 8D6 did not showblocking activity. Furthermore, five other known human FDC-specific mAbsobtained by various investigators, namely DRC-1 (Naiem, M., et al., J.Clin, Pathol. 36:167-175, 1983), 7D6 (Liu et al., J. Exp. Med.185:165-170, 1997), HJ-2 (Butch et al., Cell. Immunol. 155: 27, 1994),GP93 (Farace et al., Eur. J. Immunol. 16: 1521, 1986), and Ki-M4(Parwaresch et al., Blood 62: 585, 1983), did not inhibit the FDCcostimulatory activity in the assay.

[0061] In a separate experiment, GC-B cells were cocultured with orwithout mAb 8D6 in the presence of irradiated HK cells, CD40L, rhIL-2,and rhIL- 10. HK is a human FDC line which resembles primary FDCs in theability to rescue GC-B cells from apoptosis (Kim, H. -S, et al., J.Immunol. 155:1101-1109, 1995) and to support GC-B cell growth anddifferentiation (Choe, J., et al., J. Immunol. 157:1006-1016, 1996).GC-B cells (10⁶ cells/ml) were cocultured with irradiated HK cells(2×10⁴ cells per well; 5,000 rads) in 24-well plates with or without mAb8D6 (10 mg/ml) in the presence of CD40L (100 ng/ml), rIL-2 (10 U/ml),and rIL-10 (20 ng/ml) for 2 days, washed, and then recultured (2×10⁵cells/well) with irradiated HK cells for another 4 days. Viable cellswere counted by trypan blue exclusive assay for proliferation. Fordifferentiation, triplicate culture supernatants were harvested andpooled, and IgG concentrations were measured by ELISA as described by Liet al. (Cell. Immunol. 168:133-140, 1996). As shown in FIG. 2B, GC-Bcell proliferation and differentiation supported by HK cells wereinhibited by the addition of mAb 8D6, but not by the isotype control mAb3C8.

[0062] The cell cycle progression of the CFSE-labeled GC-B cells afteractivation by soluble CD40L was also inhibited by the addition of mAb8D6. For cell cycle analysis, GC-B cells were labeled withcarboxyfluorescein diacetate succinimidyl ester (CFSE, 5 mM/ml in PBS;Sigma Chemical Co.) at 48° C. for 10 min. Labeled GC-B cells were thencocultured with or without mAb 8D6 in the presence of irradiated HKcells, CD40L, rhIL-2, and rhIL-10, as described above. After culture for6 days, the CFSE intensity was analyzed by FACScan™. As shown in FIG.2C, 19% of GC-B cells remained in the first generation (G1) when mAb 8D6was present in the culture, as relative to 9% in the absence of mAb 8D6;16% of GC-B cells remained in the second generation (G2), compared to11% in the absence of mAb 8D6. This result indicates that mAb 8D6 exertsits inhibitory effect by delaying B cell proliferation, rather than bydirect cell killing.

EXAMPLE 4 The Growth Inhibition of Lymphoma Cell Line, L3055, by mAb 8D6and mAb 4G10.

[0063] GC-B cells undergo complex interactions with FDCs and T cells inthe course of differentiation into memory B and plasma cells. GC-B cellsfreshly isolated from tonsils are heterogeneous with respect to thestage of differentiation, mutation frequency, and Ig class (Pascual, V.,et al., J. Exp. Med. 180:329-339, 1994). To delineate the individualroles of FDCs and T cells in GC-B cell differentiation at the clonallevel, a unique experimental model was developed which employed the FDCline, HK, and a Burkitt's lymphoma cell line, L3055. L3055 cell lineresembles normal centroblasts and represents a clonal populationoriginating from the GC (Choe et al., J. Immunol. 164:5643, 2000).

[0064] Like freshly isolated centroblasts, L3055 cells underwentspontaneous apoptosis when cultured in the absence of fresh FDCs or HKcells. However, when L3055 cells (2×10⁴ cells/well) were cultured in thepresence of irradiated HK cells (2×10⁴ cells per well; 5,000 rads),L3055 cells proliferated continuously (FIG. 3A). Unlike HK cells,irradiated CD32-transfected L cells (2×104 cells per well; 5,000 rads)did not support the growth of L3055 (FIG. 3A), indicating that thegrowth-supporting activity of HK cells was not a specific feeder celleffect.

[0065] In a separate experiment, L3055 cells (2×10⁴ cells/ml) werecultured for 5 days with irradiated HK cells (2×10⁴ cells per well;5,000 rads) which had been pretreated with mAb 8D6, mAb 4G10, or acombination of mAb 8D6 and mAb 4G10 (20 μg/ml each). At the end of the5-day culture, viable L3055 cells were counted by trypan blue exclusionassay, and viable cell recovery percentages are shown in FIG. 3B. Thegrowth-supporting activity of HK cells in this coculture was decreasedby the addition of mAb 8D6 or 4G10 (FIG. 3B).

[0066] To determine whether mAb 8D6 affected apoptosis, L3055 cells (10⁴cells/ml) were cultured in the presence or absence of irradiated HKcells for 24 h. HK cells were pretreated with mAbs 8D6 or 3C8 forblocking experiments. Cells were stained with FITC-labeled annexin V andpropidium iodide, and the percentage of viable cells were calculated. Asshown in FIG. 3C, mAb 8D6 did not interfere with the capacity of HKcells to prevent spontaneous apoptosis.

EXAMPLE 5 Cloning and Expression of cDNA Encoding 8D6 Ag

[0067] As the HK cells expressed the 8D6 Ag (FIG. 1G), a cDNA expressionlibrary was prepared from the HK cells. The cDNA library was screened byin situ staining of transiently transfected COS cells with the mAb 8D6.A cDNA clone encoding the 8D6 Ag was isolated and the encoded amino acidsequence of 282 residues was set forth in SEQ ID NO: 1. The protein,named as “FDC-SM-8D6” (“SM” for signaling molecule), has a putativesignal peptide (Met1-Gly30) and a predicted transmembrane domain(Ile232-L250). There are three consensus sites (Asn126, Asn195 andAsn213) for N-linked glycosylation.

[0068] The functional activity of FDC-SM-8D6 in B cell costimulation wasanalyzed in vitro by using transfected COS cells. COS cells (2×10⁵ well)in 6-well plates were transfected with 2 mg 8D6 Ag cDNA andLipofectAMINE. After 24 h, transfected COS cells were used for coculturewith tonsillar B cells (10⁶ ml) in the presence of anti-CD40 (100ng/ml), rhIL-2 (10 U/ml), rhIL-4 (50 U/ml), rhIL-6 (20 ng/ml), andrhIL-10 (20 ng/ml) for 24 h. Activated B cells were removed from COScells, recultured in triplicate in 96-well plates in the presence of theabove cytokines, and subjected to proliferation (3 day) ordifferentiation (10 day) assays as described above.

[0069] As shown in FIGS. 4A-4C, in the cocultures withanti-CD40-activated B cells, the FDC-SM-8D6-transfected COS cellsenhanced B cell proliferation (FIG. 4A) and differentiation by inducingIgG secretion (FIG. 4B) two to seven times higher than themock-transfected COS cells. In addition, this activity was specificallyblocked by mAb 8D6 (FIG. 4C), but not by the control Ab with the sameisotype. Thus, FDC-SM-8D6 expressed by COS cells was able to provide aspecific costimulatory signal in augmenting growth and differentiationof GC-B cells.

EXAMPLE 6 Inhibition of B Cell Lymphomagenesis in vivo by mAb 8D6 andmAb4G10

[0070] Solid tumors were formed when L3055 cells (2×10⁶ cell/site) andHK cells (1×10⁶ cell/site) were subcutaneously (s.c.) inoculated intonude mice (FIG. 5B). The s.c. injected cells produced localprogressively growing tumor masses at the injection site without distantmetastasises in nude mice. There was no tumorigenesis when either L3055(FIG. 5A) or HK cells alone were injected. The failure of tumorigenesiswas not attributed to the inoculation period of 60 days. There was notumor formed if the inoculation period was longer than 4 months.

[0071] HK cell titration experiments showed that tumors could form whena lower dose of HK cells (5×10⁵ cell/site) was co-inoculated with L3055cells. When a fixed number of HK cells (1×10⁶ cell/site) were injectedwith increasing number of L3055 cells, tumor formation was observed in ashortened time frame. In contrast, no tumor was formed when increasingnumbers of L3055 cells up to 4×10⁶ cell/site were injected alone. Theseresults indicate that the lack of tumor formation when L3055 cells wereinjected alone did not result from an insufficient number of injectedcells.

[0072] FACS analysis of tumor cell surface markers were performed toidentify the origin of the tumor mass. L3055 cells express high levelsof CD38 but no CD44, while CD44 is a marker for HK cells. The resultsfrom FACS analysis showed that the tumor cells were CD20⁺CD38⁺CD44⁻,confirming that the tumor cells originated from the injected L3055cells, not from cells of murine origin. In addition,immunohistochemistry staining of tumor tissue biopsy indicated that thetumor cells were CD38⁺CD44⁻, which is identical phenotype to L3055 cellsinoculated.

[0073] To determine the effect of mAbs 8D6 and 4G10 on lymphomaformation, mAb 8D6 or mAb 4G10 was injected into mice together withL3055 cells and HK cells. The volumes of solid tumors were measured atdifferent time points. As shown in FIG. 6A, both mAb 8D6 and mAb 4G10delayed lymphoma formation. At 38 days after injection, tumors formed inthe presence of mAb 8D6 (FIG. 6C(2)) were smaller than those in thecontrol animals formed without mAb8D6 (FIG. 6C(1)). Only one tumor outof two injection sites was formed in the presence of mAb 4G10 (FIG.6C(3)). No tumor was formed when both of the mAbs were added (FIG.6C(4)). As summarized in FIG. 6B, the differences in tumor size betweenthe control group and the group with mAb 8D6 (p=0.0014) or 4G10(p=0.0023) pre-treated HK cells were statistically significant. Inaddition, the inhibitory effect of the combination of mAbs 8D6 and 4G10on tumorigenicity of L3055 cells, relative to the effect of the controlantibody, was also statistically significant effect (p=0.0001).

EXAMPLE 7 FDC-SM-8D6 Stimulates PC Generation, but Not Memory B CellProliferation

[0074] Kinetic experiments revealed that cytokines secreted by activatedT cells determined the pathway of GC-B cell differentiation. IL-4directed GC-B cells to differentiate into memory B cells, whereas IL-10steered B cells into plasma cells (PC). FDC/HK cells did not directeither pathway, but appeared to provide signals supportive of GC-B cellsproliferation in the GC.

[0075] To investigate the role of FDC-SM-8D6 in the process of GC-B celldifferentiation, mAb 8D6 (50 μg/ml) was added at the beginning ofculture of GC-B cells (1×10⁵ cell/well) and HK cells (2×10⁴ cell/well,5,000 Rad) in the presence of CD40L (100 ng/ml), IL-2 (30 U/ml), IL-4(50 U/ml) or IL-10 (50 ng/ml). After 7 days of culture, GC-B cells wereharvested for viable cell count by trypan blue exclusive assay. Viablecell recoveries were calculated as a percentage of the initial viablecell number. At the same time, cells were stained with FITC-conjugatedanti-CD20 and PE-conjugated anti-CD38 mAbs for FACS analysis. Theabsolute number of PCs and memory B cells were determined by multiplyingviable cell count with CD20⁻CD38^(hi) or CD20⁺CD38^(lo) cell frequency.The culture supernatant was harvested for measuring the Ig secretion inELISA.

[0076] In the culture containing IL-10, viable cell recovery was 920% atthe end of culture (FIG. 7B, right). Of total cells recovered, 49% werePC as determined by the number of CD20⁻CD38^(hi) cells (FIG. 7A). By day7, IgG concentration in the supernatant of the culture containing IL-10was 18.3 μg/ml (FIG. 7D). Compared to the control cultures, the additionof mAb 8D6 reduced the cell recovery from 920% to 435% (FIG. 7B) and thepercentage of PC from 49% to 24% (FIG. 7A). The absolute number of PCwas decreased by 78% when mAb 8D6 was present (FIG. 7C). The decrease ofPC number was also reflected by 61% reduction of IgG secretion if mAb8D6 was added (FIG. 7D). Such inhibitory effect was not observed in theculture containing isotype matched control mAb 3C8 which bound FDC, butwhich did not inhibit the FDC/HK cell-mediated GC-B cell growth.However, mAb 8D6 did not affect memory B cell proliferation in theculture containing IL-4 (FIGS. 7B-7D, left). These data indicate thatmAb 8D6 selectively inhibited PC generation and reduced IgG secretion inthe cultures containing IL-10.

[0077] The inhibitory effect of mAb 8D6 was observed only when mAb 8D6was added at the beginning of the cultures. When mAb 8D6 was added 3days after initiation of the culture, there was no significantinhibitory effect (FIGS. 7B-D, gray bars). These results indicate thatthe stimulation by FDC-SM-8D6 in the early stage of PC generation iscritical.

[0078] The specific function of FDC-SM-8D6 in the generation of PCs wasfurther investigated. Centrocytes were generated by culturingcentroblasts for 5 days in the presence of HK cells, CD40L, IL-2, andIL-4 in the first step of culture. In the second step of culture, IL-4was replaced by IL-10, and cells were cultured for an additional 7 days.At the end of the 7-day culture, 31% of recovered cells were determinedto be CD20⁻CD38^(hi) PC (FIG. 8). In the same experiment, 7.4 μg/ml ofIgG was detected in the culture supernatant (FIG. 9). Meanwhile, theculture containing IL-4 did not produce PC or IgG. The addition of mAb8D6 reduced viable cell number by 38% in the second step culturecontaining IL-10, whereas it did not affect cell growth in the culturecontaining IL-4 throughout. The selective inhibition of PC generation bymAb 8D6 was confirmed by 43% reduction of IgG secretion in the culturewith IL-10, but not in the culture with IL-4. These results indicatethat the target cells of FDC-SM-8D6 are PC precursors in the GC.

1 1 1 282 PRT Homo sapiens 1 Met Ser Gly Gly Trp Met Ala Gln Val Gly AlaTrp Arg Thr Gly Ala 1 5 10 15 Leu Gly Leu Ala Leu Leu Leu Leu Leu GlyLeu Gly Leu Gly Leu Glu 20 25 30 Ala Ala Ala Ser Pro Leu Ser Thr Pro ThrSer Ala Gln Ala Ala Gly 35 40 45 Pro Ser Ser Gly Ser Cys Pro Pro Thr LysPhe Gln Cys Arg Thr Ser 50 55 60 Gly Leu Cys Val Pro Leu Thr Trp Arg CysAsp Arg Asp Leu Asp Cys 65 70 75 80 Ser Asp Gly Ser Asp Glu Glu Glu CysArg Ile Glu Pro Cys Thr Gln 85 90 95 Lys Gly Gln Cys Pro Pro Pro Pro GlyLeu Pro Cys Pro Cys Thr Gly 100 105 110 Val Ser Asp Cys Ser Gly Gly ThrAsp Lys Lys Leu Arg Asn Cys Ser 115 120 125 Arg Leu Ala Cys Leu Ala GlyGlu Leu Arg Cys Thr Leu Ser Asp Asp 130 135 140 Cys Ile Pro Leu Thr TrpArg Cys Asp Gly His Pro Asp Cys Pro Asp 145 150 155 160 Ser Ser Asp GluLeu Gly Cys Gly Thr Asn Glu Ile Leu Pro Glu Gly 165 170 175 Asp Ala ThrThr Met Gly Pro Pro Val Thr Leu Glu Ser Val Thr Ser 180 185 190 Leu ArgAsn Ala Thr Thr Met Gly Pro Pro Val Thr Leu Glu Ser Val 195 200 205 ProSer Val Gly Asn Ala Thr Ser Ser Ser Ala Gly Asp Gln Ser Gly 210 215 220Ser Pro Thr Ala Tyr Gly Val Ile Ala Ala Ala Ala Val Leu Ser Ala 225 230235 240 Ser Leu Val Thr Ala Thr Leu Leu Leu Leu Ser Trp Leu Arg Ala Gln245 250 255 Glu Arg Leu Arg Pro Leu Gly Leu Leu Val Ala Met Lys Glu SerLeu 260 265 270 Leu Leu Ser Glu Gln Lys Thr Ser Leu Pro 275 280

What is claimed is:
 1. Monoclonal antibody 8D6 (ATCC#).
 2. Monoclonalantibody 4G10 (ATCC#).
 3. A functional derivative of mAb 8D6 (ATCC#),wherein said functional derivative suppresses B cell growth ordifferentiation.
 4. The functional derivative of mAb 8D6 (ATCC#),wherein said functional derivative is selected from Fab, Fab′, F(ab′)₂,a single chain antibody, or a chimeric antibody.
 5. A functionalderivative of mAb 4G10 (ATCC#), wherein said functional derivativesuppresses B cell growth or differentiation.
 6. The functionalderivative of mAb 4G10 (ATCC#), wherein said functional derivative isselected from Fab, Fab′, F(ab′)₂, a single chain antibody, or a chimericantibody.
 7. A pharmaceutical composition comprising mAb 8D6 and apharmaceutically-acceptable carrier.
 8. A pharmaceutical compositioncomprising a functional derivative of mAb 8D6 (ATCC#) and apharmaceutically-acceptable carrier, wherein said functional derivativesuppresses B cell growth or differentiation.
 9. The pharmaceuticalcomposition of claim 8, wherein said functional derivative is selectedfrom Fab, Fab′, F(ab′)₂, a single chain antibody, or a chimericantibody.
 10. A pharmaceutical composition comprising mAb 4G10 (ATCC#).and a pharmaceutically-acceptable carrier.
 11. A pharmaceuticalcomposition comprising a functional derivative of mAb 4G10 (ATCC#) and apharmaceutically-acceptable carrier, wherein said functional derivativesuppresses B cell growth or differentiation.
 12. The pharmaceuticalcomposition of claim 11, wherein said functional derivative is selectedfrom Fab, Fab′, F(ab′)₂, a single chain antibody, or a chimericantibody.
 13. A pharmaceutical composition comprising (1) apharmaceutically-acceptable carrier, (2) mAb 8D6 (ATCC#) or a functionalderivative of mAb 8D6, and (3) mAb 4G10 or a functional derivative ofmAb 4G10.
 14. A method of treating a subject suffering a pathologicalcondition characterized by abnormal growth or differentiation of Bcells, comprising administering to said subject a therapeuticallyeffective amount of mAb 8D6 or a functional derivative thereof.
 15. Themethod of claim 14, wherein said condition is lymphoma.
 16. The methodof claim 14, wherein said condition is multiple myeloma.
 17. The methodof claim 14, wherein said condition is an autoimmune disease.
 18. Themethod of claim 14, wherein said functional derivative is selected fromFab, Fab′, F(ab′)₂, a single chain antibody, or a chimeric antibody. 19.A method of treating a subject suffering a pathological conditioncharacterized by abnormal growth or differentiation of B cells,comprising administering to said subject a therapeutically effectiveamount of mAb 4G10 or a functional derivative thereof.
 20. The method ofclaim 19, wherein said condition is lymphoma.
 21. The method of claim19, wherein said condition is multiple myeloma.
 22. The method of claim19, wherein said condition is an autoimmune disease.
 23. The method ofclaim 19, wherein said functional derivative is selected from Fab, Fab′,F(ab′)₂, a single chain antibody, or a chimeric antibody.
 24. A methodof treating a subject suffering a pathological condition characterizedby abnormal growth or differentiation of B cells, comprisingadministering to said subject a therapeutically effective amount of (1)mAb 4G10 or a functional derivative thereof, and (2) mAb 8D6 or afunctional derivative thereof.
 25. A hybridoma cell line, having theATCC deposit number ______.
 26. A hybridoma cell line, having the ATCCdeposit number ______.